Interference and blood sample preparation for a pyruvate enzymatic assay

BACKGROUND: To assess the severity of circulatory failure, a pyruvate enzymatic assay was performed on whole blood using lactate dehydrogenase to catalyze the conversion of pyruvate to lactate. We investigated factors related to blood sample collection and preparation that might influence the results, including the timing of blood deproteinization, temperature of sample storage, and hemolysis. METHOD: A total of 25 whole blood specimens were collected for this study. Each sample was divided into 2 parts: one stored at room temperature (RT) and another kept on ice. The samples were deproteinizied by using 8% perchloric acid (PCA) at varying times after collection; the first deproteinization was immediately after the blood was drawn (0 h), then at 1 h intervals for 6 h and also in samples kept overnight. The supernatant samples were analyzed soon after deproteinization using a COBAS Centrifugal Analyzer. In another set of samples, the blood was immediately deproteinized, and the supernatants were stored at RT and 4 degrees C and assayed for pyruvate at varying times, as above. Finally, the effect of hemolysis on the blood pyruvate enzymatic assay was also evaluated. RESULTS: When samples were stored at RT, pyruvate levels remained constant until the third h after deproteinization, when there was an approximately 13.3% increase in pyruvate concentration. When whole blood samples were kept at 4 degrees C before deproteinization, pyruvate levels were significantly reduced over time, ranging from 37.8% to 62.2% (paired t test showed a significant mean difference, P < 0.001). No significant differences in pyruvate concentration were observed in supernatant stored at either RT or 4 degrees C. Hemolysis caused a 33.7% increase in the pyruvate concentration, equivalent to 0.18 mg pyruvate per gram per deciliter of hemoglobin. CONCLUSIONS: For a pyruvate enzymatic assay, keeping a whole blood sample at RT will not cause a significant difference in the pyruvate level as long as the sample is immediately deproteinized. Whole blood samples should not be stored in an ice bath for transport, nor should hemolyzed samples be used for a blood pyruvate enzymatic assay.     

动脉血气分析结果可靠的血样放置时限

目的 确定动脉血样采集后得到准确可靠血气分析检测结果 的标本放置时限.方法 选择择期行口腔颌面外科手术患者40例,全麻诱导后气管插管,进行机械通气后30min,采集桡动脉血液,进行血气分析,红细胞压积、血红蛋白、血糖、乳酸、电解质测定.检测时点分别为采血后即刻(T0)、采血后15min(T1)、采血后30min(T2)、采血后45min(T3)、采血后60min(T4)、采血后90min(T5)、采血后120min(T6)、采血后180min(T7)和采血后240min (T8).结果 从T4开始pH和PaO2比T0明显降低,PaCO2明显增高,P＜0.05.HCO3-、TCO2和BE在T7和T8时刻显著降低.SaO2在各时点无明显变化.从T4开始红细胞压积、血红蛋白和乳酸比T0明显增高,P＜0.05.从T4开始血糖比T0明显降低,P＜0.05.Na+浓度略降低,K+浓度略增高,但均无统计学意义,P＞0.05.Ca2+浓度在各时点无明显差异.结论 动脉血采集常温放置45min后血气分析结果 仍然准确可靠,具有临床指导意义.     

Stability of human blood serum aminoacids after storage at different pH and temperature conditions

The stability of the aminoacids in human blood serum ultrafiltrates and in an aminoacid standard solution was investigated under different pH and storage conditions. At close to neutral pH values the aminoacid concentrations remained constant for at least 12 months at -50 degrees C (n = 6), except for lysine. With acid but particularly with alkaline conditions a time- and temperature-dependent decrease was observed for glutamine and asparagine with concomitant increases of glutamate and aspartate. Similar, but less prominent alterations were noted for the concentrations of methionine, glycine, tyrosine, histidine, arginine and ornithine. Almost independent of pH, there was an effect of temperature; after 24 h at 55 degrees C a significant increase of several per cent in a number of serum aminoacid concentrations was observed, presumably due to the hydrolysis of small proteins and peptides. For the purpose of aminoacid analysis it is recommended that samples be stored deproteinized, deep-frozen and at neutral pH.     

Effect of blood collection and processing on radioimmunoassay results for apolipoprotein A-I in plasma

We studied the effects of different procedures of blood collection and processing on quantification of apolipoprotein A-I (apoA-I) by radioimmunoassay. ApoA-II and apolipoproteins of low- and very-low-density lipoproteins did not cross react in the assay. Analytical recovery of apoA-I at different doses was complete. ApoA-I concentration in pooled human plasma was stable for as long as a year stored at -70 degrees C. Inter- and intra-assay CVs averaged 7% and 5%, respectively. We collected blood from 20 subjects into tubes containing EDTA alone or EDTA with antiproteolytic agents, then separated the plasma either immediately or after 3 h at 4 degrees C. We tested various formulations of antibacterial, antiproteolytic, and anti-oxidant agents added to plasma, measuring apoA-I concentrations either within 24 h of blood collection or after storage of plasma for 6 weeks at -70 degrees C. No significant difference in the concentrations of apoA-I was found in these specimens, regardless of the conditions studied. We conclude that addition of protective agents other than EDTA is not necessary during blood collection or specimen processing for reliable quantification of apoA-I in fresh or frozen human plasma.     

Can fluoride-oxalate and sodium citrate stabilise homocysteine levels after blood collection?

The concentration of homocysteine (Hcy) rises rapidly after the collection of blood. This feature requires blood to be collected into the anticoagulants EDTA or heparin and the plasma to then be immediately separated; alternatively, the blood may be kept on ice and centrifuged within 1 hour. The use of chemical preservatives has been proposed as a means of stabilising Hcy levels in whole blood after collection. The objective of this study was to determine whether the commonly available fluoride-oxalate (Fl-Ox) and sodium citrate (Na-Cit) containers could stabilise Hcy levels in blood. Our results showed that when blood was collected into potassium EDTA (K-EDTA) tubes, Hcy levels rose from initial levels, on standing at room temperature (approximately 25 degrees C), by an average of 21% after 3 hours and 32% after 5 hours. The initial Hcy levels of blood collected into Fl-Ox and Na-Cit containers, however, were lower, at averages of 89% and 91%, respectively, compared to that of the same samples when collected into K-EDTA tubes. Hcy in these samples subsequently rose on standing, and after 5 hours was, on the average, 10 and 13% higher, respectively, compared with the initial levels in K-EDTA tubes. We conclude that Fl-Ox and Na-Cit do not stabilise Hcy in blood after collection and should not be used as preservatives.     

Storage of G-CSF-mobilized granulocyte concentrates

BACKGROUND: Current standards limit granulocyte storage to 24 hours. Since G-CSF inhibits granulocyte apoptosis, it may be possible to store G-CSF-mobilized granulocytes for longer periods while maintaining cell viability and function. However, G-CSF mobilization increases the yield of granulocytes several times, and the resulting higher cell concentrations may diminish viability during storage and significant levels of pyrogenic cytokines may be produced. STUDY DESIGN: Ten granulocyte donors were given dexamethasone (8 mg PO), G-CSF (5 microg/kg SQ), or both and on the next day granulocyte concentrates were collected using a blood cell separator. Component cell counts, cell viablilities, pH, and IL-1beta, IL-6, IL-8 and TNF levels were measured at 2 to 4 (2), 20 to 28 (24), and 44 to 52 hours (48 hours). RESULTS: Significantly more granulocytes were collected when donors were given G-CSF (4.2 +/- 2.3 x 10(10)) or G-CSF plus dexamethasone (6.4 +/- 2.5 x 10(10)) compared with that collected with dexamethasone alone (2.2 +/- 1.2 x 10(10)); p = 0.03 and p = 0.002, respectively. Storage had little effect on WBC count. Slight but significant increases in IL-1beta and IL-8 occurred after 24 and 48 hours as compared to the levels at 2 hours' storage. Levels of IL-6 and TNF did not change. The pH dropped significantly with time in granulocytes mobilized with each regimen. Granulocytes mobilized with G-CSF plus dexamethasone were acidic immediately after collection, and pH was below 6.0 after 24 hours. To assess the effect of cell concentrations on pH, serial dilutions were performed on 13 granulocyte concentrates in autologous plasma prior to storage. The pH remained above 7.0 only when dexamethasone-mobilized granulocytes were diluted 1-in-8 and when the G-CSF plus dexamethasone-mobilized granulocytes were diluted 1-in-16. CONCLUSIONS: To optimize storage pH, mobilized granulocyte concentrates require a 1-in-8 to 1-in-16 dilution, which is operationally impractical. Clinical-grade granulocyte preservative solutions are needed to maintain pH during storage.     

Optimal conditions for collection of blood samples for assay of α2-macroglobulin trypsin complex-like substance (MTLS)

We have developed an enzyme-linked immunosorbent assay (ELISA) for the specific quantification of α2-macroglobulin-trypsin complex-like substance (MTLS). To exclude artifacts in measured values of MTLS, the conditions for collection of blood samples are critical. In the present study, we have determined the optimal conditions for blood collection and investigated the role of MTLS as a clinical tool for diagnosis in pancreatitis. Results obtained are as follows: (1) MTLS levels of all sera were more than 10-fold higher than the corresponding plasma; (2) MTLS levels of heparinized plasma were the lowest among plasma with three anticoagulants (sodium citrate, sodium EDTA and heparin); (3) some kinds of blood collection tubes containing heparin were not suitable for the sampling; (4) MTLS values of plasma obtained by blood collection tubes containing Trasylol® and sodium EDTA were demonstrated more stable and lower than those obtained by heparin tubes; and (5) under these conditions, we can exclude elevation of MTLS values caused by inappropriate blood sampling and find the time course of the elevation reflecting clinical course of a patient with acute pancreatitis and a patient after endoscopic retrograde cholangiopan-creatography (ERCP). The optimal conditions for collection of blood samples were as follows. Blood sampling should be performed by blood collection tubes containing Trasylol® (50 μl/ml blood) and sodium EDTA (1.5 mg/ml blood). The samples were immediately stored at 4°C and centrifuged at 3,000 rpm for 15 min. The plasma were stored in plastic tubes at 4°C until assayed. © 1996 Wiley-Liss, Inc.     

Effect of blood collection and processing on radioimmunoassay results for apolipoprotein B in plasma

We studied the effects of different blood collection and processing procedures on quantification of apolipoprotein (apo) B by radioimmunoassay. High-density lipoprotein subfractions HDL3 and HDL2 and isolated apoA-I did not cross-react in the assay. Analytical recovery of apoB at different doses of very-low- and low-density lipoproteins were complete. Inter- and intra-assay coefficients of variation (CVs) averaged 7.4% and 6.0%, respectively. Blood from 20 subjects was collected into tubes containing EDTA alone or EDTA with antiproteolytic and antioxidant agents; one half of each plasma was separated immediately, half after 3 h at 4 degrees C. Regardless of the addition of protective agents or the time difference in separating plasma from other blood elements, freezing plasma at -70 degrees C decreased apoB content a similar amount, an average of 6.8%. This loss of apoB immunoreactivity was not related to apoB content in fresh plasma. Analysis of variance showed no differential effect on apoB content by the various additions to whole blood or plasma. No additional apoB content was lost in once-frozen aliquots of three human plasma pools during storage at -70 degrees C for up to 18 months. We conclude that concentrations of apoB in human plasma can be measured reliably after long-term storage, although the absolute value may decrease slightly as a result of freezing.     

Stability of mycophenolic acid and glucuronide metabolites in human plasma and the impact of deproteinization methodology

BACKGROUND: In recent years there is growing interest in therapeutic drug monitoring of mycophenolic acid (MPA) and its glucuronide metabolites MPAG and AcMPAG. Like other acyl glucuronide metabolites, AcMPAG has a limited stability, but this aspect has received little attention. METHODS: Plasma sample deproteinization with perchloric acid 2 M (method A) was compared to metaphosphoric acid 15% (method B). Stability of MPA, MPAG and AcMPAG in acidified and non-acidified plasma stored at room temperature, 4 degrees C, -20 degrees C and -80 degrees C was assessed over short and long time intervals using HPLC-UV methodology. RESULTS: The area ratio of AcMPAG/IS on spiked plasma at pH 2.5 with method A was 63% of the respective ratio in water, in contrast to 102% with method B, suggesting partial deconjugation and/or incomplete release of AcMPAG from proteins with method A. At room temperature, AcMPAG concentrations in both whole blood and non-acidified plasma decreased significantly after 2-5 h. MPA, MPAG and AcMPAG concentrations remained stable in acidified plasma stored at -20 degrees C and -80 degrees C, but not longer than 5 months after collection. CONCLUSIONS: It is concluded that adequate sample collection, storage measures and deproteinization methods should be applied in order to avoid deconjugation and hence underestimation of MPA, MPAG and AcMPAG concentrations.     

Stability studies of twenty-four analytes in human plasma and serum

BACKGROUND: The stability and stoichiometric changes of analytes in plasma and serum after prolonged contact with blood cells in uncentrifuged Vacutainer tubes were studied. METHODS: We simultaneously investigated the stability of 24 analytes (a) after prolonged contact of plasma and serum with blood cells and (b) after immediate separation of plasma and serum (centrifuged twice at 2000g for 5 min). We verified biochemical mechanisms of observed analyte change by concomitant measurement of pH, PCO(2), and PO(2). Hemolysis was qualitatively and semiquantitatively assessed. All specimens were maintained at room temperature (25 degrees C) and analyzed in duplicate 0.5, 4, 8, 16, 24, 32, 40, 48, and 56 h after collection. Statistically significant changes from the 0.5 h mean were determined using repeated-measures ANOVA. The significant change limit was applied to determine clinically significant changes in measured analytes. RESULTS: Fifteen of 24 analytes in plasma and serum maintained in contact with cells showed clinically relevant changes, with the degree of change more pronounced in most plasma specimens. All analytes in plasma and serum immediately separated from cells after collection were stable. CONCLUSION: Storage of uncentrifuged specimens beyond 24 h caused significant changes in most analytes investigated because of (a) glucose depletion and Na(+),K(+)-ATPase pump failure; (b) the movement of water into cells, causing hemoconcentration; and (c) leakage of intracellular constituents and metabolites. Immediate separation of plasma or serum from cells provides optimal analyte stability at room temperature. When prolonged contact of plasma or serum with cells is unavoidable, use of serum is recommended because of the higher instability of plasma analytes.     

Impact of short-term storage of plasma samples on quantitation of ultra-low levels of interleukin-6

ObjectiveTo quantitate plasma interleukin-6 (IL-6) levels in healthy individuals and to clarify how these levels are affected by blood sample handling procedures during short-term storage.MethodsEthylenediaminetetraacetic acid (EDTA)-treated plasma samples were simultaneously collected from 14 healthy individuals and stored on ice prior to analysis of the IL-6 levels. White blood cells (WBCs), red blood cells, and platelets were counted immediately after blood collection. IL-6 levels were analyzed every 30 minutes using a commercial electrochemiluminescence immunoassay.ResultsCorrelation coefficients between plasma IL-6 levels and WBC counts ranged between 0.605 and 0.554, higher than those for other cell types. The lowest IL-6 value in healthy individuals was estimated at 0.04 pg/mL and the mean values remained under 2 pg/mL over time.ConclusionAnalysis of IL-6 levels in EDTA-treated plasma samples centrifuged within 1 hour and stored on ice can be performed within 90 minutes of short-term storage if the analytical method has a sensitivity in the range of 10 fg/mL.     

Assessment of the stability of N-terminal pro-brain natriuretic peptide in vitro: implications for assessment of left ventricular dysfunction.

Plasma concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP) are raised in patients with left ventricular dysfunction. Measurement of this peptide has a potential diagnostic role in the identification and assessment of patients with heart failure. The stability of this peptide over time periods and conditions pertaining to routine clinical practice has not been reported previously. Blood samples were obtained from 15 subjects. One aliquot was processed immediately, and the remaining portions of the blood samples were stored for 24 h or 48 h at room temperature or on ice prior to processing. Plasma concentrations of NT-proBNP were measured with a novel immunoluminometric assay developed within our laboratory. Mean plasma concentrations of NT-proBNP were not significantly different whether blood samples were centrifuged immediately and stored at -70 degrees C or kept at room temperature or on ice for 24 h or 48 h. The mean percentage differences from baseline (reference standard) were +5.2% (95% confidence interval +18.2 to -7.8%) and +0.8% (+15.2 to -13.7%) after storage for 24 h at room temperature or on ice respectively, and +8.9% (+24.2 to -6. 5%) and +3.2% (+15.1 to -0.9%) for storage for 48 h at room temperature or on ice respectively. Pearson correlation coefficients for baseline NT-proBNP concentrations compared with levels at 48 h at room temperature or on ice were r=0.89 and r=0.83 respectively (both P<0.0001). Thus NT-proBNP extracted from plasma samples treated with EDTA and aprotinin is stable under conditions relevant to clinical practice.     

Ionized calcium: whole blood, plasma or serum?

OBJECTIVES: To determine an optimal specimen type to be used for measurements of ionized calcium (iCa) so that it applies properly to the reference interval. Also to determine the validity of the pH correction that is applied to iCa measurements. METHODS: A reference interval study of normal volunteers was performed using four sample types namely balanced heparin (BH) whole blood, lithium heparin (LH) whole blood, plasma and serum. The sample was treated in an anaerobic fashion and analyzed at 0 and 40 minutes after venipuncture. The effect of pH correction as well as analysis time after collection was also studied. RESULTS: The mean iCa was the highest in BH-treated whole blood when measured immediately. However, it was slightly lower at 40 min after collection (p < 0.001). In contrast, there did not appear to be a significant difference in results when LH-treated whole blood was analyzed at 0 or 40 min. The reference interval for serum was similar to that of whole blood. The reference interval for plasma was dramatically lower than whole blood and plasma. The reference intervals for pH adjusted ionized calcium (iCa-adj) were dramatically lower than those from all specimens without adjustment. The reason for this was that the reference interval for pH in this study had a strong alkaline bias on one instrument and a strong acidic bias on the other. CONCLUSIONS: The sample of choice for ionized calcium analysis appears to be whole blood with either BH or LH. For the LH specimen, there is no significant change over 40 min whereas there is significant change for the BH specimens (-0.030 mmol/L, p < 0.0001). iCa-adj should not be used unless (i) very strict attention is paid to standardization of both the calcium and the pH and (ii) there is a very good reason to believe that the patients' pH is normal at 7.4.     

Stability of prothrombin time and activated partial thromboplastin time tests under different storage conditions

Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are common laboratory tests that are useful in the diagnosis of coagulation disorders and monitoring anticoagulant therapy. Recent expansions in the outreach laboratory services at our institution prompted us to investigate the shipping limitations for some tests, including PT and aPTT. Although we followed NCCLS guidelines for the collection of blood specimens, we observed falsely elevated PT and aPTT values due to the different storage conditions. The objective of this study is to determine the effect of conditions and duration of storage on PT and aPTT tests using plasma and whole blood samples, respectively. For this study, 36 plasma samples with normal and prolonged PT and aPTT were exposed to different storage conditions. Blood was centrifuged immediately and plasma was stored at room temperature (RT), refrigerated at 4°C, or frozen at −20°C. The samples were analyzed at 0 h and repeated at 6, 12 and 24 h under various conditions. Although statistically significant differences were observed for plasma samples for normal PT tests after 12 h at refrigerated and frozen storage conditions, the differences would not change the clinical interpretation of the results. On the other hand, samples stored refrigerated or at RT showed significant differences for aPTT at 24 h. These differences would change clinical interpretation, especially for samples with normal or near normal aPTT times. Interestingly, aPTT was significantly higher for samples stored frozen when compared to refrigerated and RT conditions at 6 h. Similar patterns were also observed on ten whole blood samples with normal PT and aPTT values. In conclusion, either plasma or whole blood samples can be accepted for PT testing up to 24 h and for aPTT testing up to 12 h only, when transported either at RT or at 4°C.     

Suitability of collection tubes with separator gels for collecting and storing blood samples for therapeutic drug monitoring (TDM).

In this study, we present significant changes occurring in serum drug concentrations while using blood collection tubes that contain a barrier gel. This report also contains results with antidepressant drugs, which have not been studied before with human samples. The drug concentrations were measured either with high performance liquid chromatography (HPLC) or fluorescence polarization immunoassay (FPIA). The results show that gel tubes are suitable for blood collection for antiepileptic, antibiotic, asthma and cardioactive drug measurements, since only slight adsorption was seen (0-5%). However, the studied tubes are not suitable for blood collection of antidepressants nor benzodiazepines, because the adsorption can be 5-30%. The adsorption was even higher (up to 40%) when samples were stored for 24 h after centrifugation in gel tubes. When the centrifugation step was performed after storage the effect of the barrier gel was lower (only 0-13%). Antidepressant drug measurements performed from patient specimens collected in the studied gel tubes and stored for 3 h showed <10% adsorption of the studied drugs. After 24 h storage time, concentrations of all analysed drugs decreased even more: adsorbed amount of drugs were about 5-20%. The studied gel tubes are proposed to be satisfactory for blood collection for antidepressant drug measurements if separation step is performed within 3 h after blood clotting. With the spiked samples the adsorption to barrier gel was higher, so it seems that adsorption is faster when drugs are not highly bound to serum proteins.     

Measurement of folate in fresh and archival serum samples as p-aminobenzoylglutamate equivalents

BACKGROUND: The development of accurate and precise folate assays has been difficult, mainly because of folate instability. Large interassay and interlaboratory differences have been reported. We therefore developed a serum folate assay that measures folate and putative degradation products as p-aminobenzoylglutamate (pABG) equivalents following oxidation and acid hydrolysis. METHODS: Serum was deproteinized with acid in the presence of 2 internal calibrators ([13C2]pABG and [13C5]5-methyltetrahydrofolate). 5-Methyltetrahydrofolate and other folate species in serum were converted to pABG by oxidation and mild acid hydrolysis. pABG and its internal calibrators were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The limit of quantification was 0.25 nmol/L, and the assay was linear in the range 0.25-96 nmol/L, which includes the 99.75 percentile for serum folate concentrations in healthy blood donors. Within- and between-day imprecision was < or = 5%. We detected no residual folate in serum samples after sample preparation. Folate concentrations in fresh serum samples obtained with the pABG assay and with a microbiologic assay showed good agreement (r = 0.96). In stored samples containing low folate concentrations due to folate degradation, the pABG assay yielded substantially higher folate concentrations than the microbiologic assay. CONCLUSIONS: The pABG assay combines automated sample preparation with LC-MS/MS analysis. It allows measurement of folate not only in fresh samples of serum/plasma but also in stored samples in which the folate has become oxidized and degraded to an extent that it cannot be assayed with traditional folate assays.     

Effects of propranolol and verapamil on plasma ionized calcium and parathyroid hormone in short-term intense isokinetic leg exercise

Summary. Physical exercise, beta-adrenergic stimulation and calcium channel blockade can affect calcium homeostasis. The present study investigated, in eight healthy males, the effects of orally administered propranolol or verapamil during a 2-min maximal, isokinetiC., leg exercise. Immediately after exercise the plasma ionized calcium concentrations were increased, in control and drug tests, by 5–6%, and within 5 min of recovery they were almost returned to baseline. Serum parathyroid hormone (PTH) concentrations were unchanged at termination of exercise, but they increased during the first 5 min of recovery, coincident with the decline in calcium concentrations, which, however, were still elevated. Neither verapamil nor propranolol selectively changed basal or exercise plasma ionized calcium or serum PTH concentrations. Muscle strength, blood pH, lactate concentrations and plasma volume changes were not affected by any drug. Verapamil did not have any specific effect on the concentrations of plasma magnesium, phosphate, potassium or sodium while propranolol increased the concentrations of plasma potassium and decreased those of phosphate during exercise as well as recovery.     

Effects of storage and handling conditions on concentrations of individual carotenoids, retinol, and tocopherol in plasma

We investigated the effects of storage and handling on measured values for carotenoids, retinol, and tocopherol in plasma. We found no significant differences in the concentrations of these analytes measured in plasma samples that were frozen immediately after separation as compared with replicate samples maintained at room temperature in the dark for 24 h. Analytes were stable in solvents for at least 18 h at 23 degrees C after extraction. Purging samples with nitrogen gas before freezing had no detectable beneficial effects. All analytes were stable in plasma stored at -70 degrees C for at least 28 months or at -20 degrees C for five months. By 15 months the concentrations of carotenoids were significantly less (P less than 0.05) in plasma stored at -20 degrees C than in plasma stored at -70 degrees C, while retinol and tocopherol concentrations were not significantly different. Concomitant with the decrease in carotenoids was the appearance of unidentified peaks in the ultraviolet. Adding ascorbic acid or butylated hydroxytoluene antioxidants to the precipitating solvent did not alter the losses of carotenoids or alter the appearance of unidentified peaks. Under appropriate conditions, plasma carotenoids, retinol, and tocopherol are stable for more than two years.     

Effects of some pre-analytical conditions on the measurement of homocysteine and cysteine in plasma.

The association of hyperhomocysteinemia and hypercysteinemia with the risk of arterial and venous thrombosis is well documented. While it is known that standardized pre-analytical conditions are necessary for reliable measurement of plasma total homocysteine, the effects of pre-analytical conditions on cysteine measurement are less well known. The aim of this study was to evaluate the effects of pre-analytical conditions on the measurement of homocysteine and cysteine. We observed that the concentration of total homocysteine in plasma increased significantly with time (38% after 6 h), whereas total cysteine decreased (5% after 2 h) when blood anticoagulated with ethylenediaminetetraacetic tripotassium salt was kept at room temperature. These changes were minimized when acidic citrate dextrose was used as an anticoagulant and were abolished when blood samples were immediately placed on crushed ice, independently of the anticoagulant. Storage of plasma for 72 h at room temperature induced a small (approximately equal to 6%), but significant, decrease in cysteine when blood was collected in ethylenediaminetetraacetic tripotassium salt. In contrast, homocysteine was stable in plasma for 72 h, independently of the anticoagulant used. In conclusion, if blood samples for plasma total homocysteine and cysteine measurement cannot be kept on ice, they should be collected in acidic citrate dextrose to minimize the artifactual changes.     

Use of a sulfate: hydrogen sulfate buffer to preserve urine for steroid analysis.

1. HCI, commonly used to preserve urine for steroid analysis, cannot safely be supplied to out of town patients. 2. An equimolar mixture of Na2SO4 and NaHSO4 is a safe alternative which may be used to protect specimens en route to the laboratory. 3. Addition of bisulfate buffer to urine (20 g/l) reduced the pH to 2.0-3.5 (31 specimens; 24 hr urine volumes, 560-3320 ml). 4. Little change in pH was observed when buffered urine (12 specimens) was kept at 30 degrees for 14 days (initial pH 2.62 +/- 0.38 (mean +/- standard deviation); PH after 14 days, 2.59 +/- 0.48). 5. Analysis of the creatinine, 17-oxosteroids, 17-oxogenicsteroids, estrogens, pregnanediol, androsterone + etiocholanolone and estriol in urine samples acidified to pH 2-3 with HCl, and in the same samples acidified with buffer, gave results which were indistinguishable from each other. 6. Almost no change in steroid and creatinine concentrations was observed in samples of buffered urine kept for 10 days at room temperature (20-26 degrees).